- Email: [email protected]
A unique point mutation in the androgen receptor gene in a family with complete androgen insensitivity syndrome*
Vol. 58, No.4, October 1992
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright 4:) 1992 The American Fertility Society
A unique point mutation in the androgen receptor gene in a family with complete androgen insensitivity syndrome*
Craig R. Sweet, M.D.t;!: Mohammad A. Behzadian, Ph.D.t§ Paul G. McDonough, M.D.tll Medical College of Georgia, Augusta, Georgia
Objective: To further delineate the diversity of genetic alterations in the gene coding for the androgen receptor in individuals with the androgen insensitivity syndrome and to increase our understanding of the disease at the molecular level. Design: This was a prospective study in which genomic deoxyribonucleic acid (DNA) from individuals with androgen insensitivity were examined through the polymerase chain reaction and DNA sequencing analysis. Patients: Eleven complete and four individuals with partial androgen insensitivity syndrome were examined. Results: Exons two through eight were grossly intact in all study subjects. Nucleotide sequence analysis revealed that three of three related family members with complete androgen insensitivity had the same guanine to adenine base substitution in exon five of the steroid-binding domain. Conclusion: The subsequent alanine to threonine amino acid conversion may have resulted in a configurational change of the androgen receptor protein leading to complete androgen insensitivity. This precise alteration has not been previously identified in the human androgen receptor gene in patients with the androgen insensitivity syndrome. Fertil Steril 1992;58:703-7 Key Words: Androgen insensitivity syndrome, point mutation
Androgens act through the human androgen receptor (hAR) to direct sexual differentiation and maintenance of the male phenotype with normal testicular exocrine function. Genetic rearrangements in the hAR gene may be responsible for a wide spectrum of functional abnormalities in the hAR resulting in the androgen insensitivity syndrome (AlS) (1). These abnormalities range from complete AlS
Received February 25, 1992; revised and accepted June 19, 1992. * Presented at the 38th Annual Meeting of the Society of Gynecologic Investigation, San Antonio, Texas, March 20 to 23, 1991. t Department of Obstetrics and Gynecology. :j: Present address: Women's Health Care and Reproductive Services, Fort Myers, Florida. § Department of Anatomy. II Reprint requests: Paul G. McDonough, M.D., Division ofReproductive Endocrinology, Medical College of Georgia, Augusta, Georgia 30912-3360. Vol. 58, No.4, October 1992
wherein 46,XY phenotypic females have a blind vaginal pouch and intra-abdominal or inguinal testes to partial AlS with genital ambiguity, hypospadias, or defects in spermatogenesis with azoospermia. The localization of the gene for the hAR to Xql1-12(2, 3) and the isolation of the complementary deoxyribonucleic acid (cDNA) in 1988 set the stage to study the molecular pathology of AlS in humans (4-6). The hAR gene, including introns, is >90 kb in length, although the coding region is divided into eight exons that are <3 kb in combined length (Fig. 1) (7-9). The hAR binds with androgens forming the androgen-hAR complex. This complex then binds to specific target genes that are responsible for the expression of the male phenotype and reproductive capabilities. Exon one codes for the N-terminal hAR domain and is the largest of the eight exons. It is hypothesized that the N-terminus Sweet et a1.
Point mutation in the androgen receptor gene
703
hAR Gene > 90 kb
I
I
•
I II eDNA 2757 bp
c
Amino Terminus Domain 1613 bp
Exon1
2345678
DNA Binding Domain 269bp
I
Steroid Binding Domain 875 bp
Protein 619 AA
Figure 1 Schematic representation of the X -linked hAR gene organization.
modulates transcription once the androgen-hAR complex is bound to the target gene DNA (9). The highly conserved DNA-binding domain codes for the two zinc finger motifs and is located in exons two and three. These areas may be involved in the recognition of the target genes in addition to stabilizing the androgen-hAR-DNA interaction (7). The remaining exons four through eight code for the androgen-binding domain. This region of the hAR gene may also be involved in unmasking the DNA-binding domain or the transcriptional activation of the target genes (8, 10). The aim of this research was to further characterize the molecular rearrangements in the hAR gene and expand our knowledge of the spectrum of molecular alterations responsible for AIS. MATERIALS AND METHODS Study Subjects
This research was approved by the Human Assurance Committee of the Medical College of Georgia. Informed consent was obtained in accordance with the Medical College of Georgia guidelines for Clinical Investigation. The subject population included sporadic (n = 6) and a family (n = 5) of complete AIS patients in addition to a sporadic (n = 1) and a family (n = 3) partial AIS individuals for a total study population of 15. The complete AIS family, shown in Figure 2, has been previously discussed (11, 12). A normal fertile male and the published normal DNA sequence served as controls (9). First-degree relatives were also examined when available and indicated. The complete AIS patients were characterized by 46,XY G-banded karyotypes, normal female external genitalia, and breast development, a blind vaginal pouch, sparse axillary and pubic hair, intra-abdominal or inguinal testes, and blood testosterone con704
Sweet et al.
Point mutation in the androgen receptor gene
centrations in the normal male range or greater. The partial AIS patients had 46,XY G-banded karyotypes with genital ambiguity and intra-abdominal or labioscrotal testes with a quantitative or qualitative deficiency in their hAR-androgen binding. Preparation of Genomic DNA and Oligonucleotide Primers
The genomic DNA was isolated from leukocytes, transformed leukocytes, or fibroblast cells (13). For each exon two through eight, two oligonucleotide primers were specifically designed to anneal to the flanking intron segments and were synthesized on the DNA synthesizer Gene Assembler Plus (Phamacia, Piscataway, NJ). The primers were 22 to 29 nucleotides in length and were similar to those used by Lubahn et al. (9). Polymerase Chain Reaction (PCR)
Polymerase chain reactions were performed in a final volume of 100 tiL containing buffer, 0.1 tIg of genomic DNA, 1.5 tiM MgCI2 , 200 tiM of each deoxynucleotide triphosphate (2'-deoxyadenosine 5'-triphosphate, 2'-deoxycytidine 5'-triphosphate, 2'deoxyguanosine 5'-triphosphate, and 2'-deoxythymidine 5'-triphosphate), 1.5 U of Taq DNA polymerase, and 1.0 tiM of each flanking primer (GeneAmp Kit; Perkin-Elmer/Cetus Instruments, Norwalk, CT). The PCR reaction was performed in a DNA Thermal Cycler (Perkin-Elmer/Cetus Instruments). The DNA was first denatured at 96°C, the primers annealed at 55 to 58°C, depending on the exon being examined, and the DNA extension was achieved at 72°C; each of these steps lasted 5 minutes. The reaction then continued at the same temperatures for 1 minute at each step for a total
Figure 2 Pedigree of the complete AIS family showing X-linked inheritance pattern. Family members A, B, C, D, E, F, and G were studied with PCR and family members A, E, and F undergoing nucleotide sequence analysis. Il!il are affected 46,XY; II!i are affected and sequenced in both orientations; 0 are obligate heterozygotes 46,XX (11). (Reprinted by permission of the publisher, The American Fertility Society, Birmingham, AL.)
Fertility and Sterility
of 30 cycles. At the completion of the 30 cycles, an additional 5-minute incubation at 72°C allowed for the completion of partially synthesized strands. The PCR products were examined on an electrophoretic agarose gel, and the respective lengths of the segments estimated using known DNA size markers (Bethesda Research Laboratories, Gaithersburg, MD). These segments ranged from 250 to 480 base pairs (bps) in length and were compared with the normal fertile male control and previously published data (9). Asymmetric PCR
The asymmetric PCR was performed to synthesize single-stranded DNA (ssDNA) for nucleotide sequence analysis. An aliquot of 1 ILL of the first PCR product was added to only a single primer and other constituents as described with the symmetric PCR. Fifty PCR cycles were performed to maximize the synthesis of the ssDNA. Dideoxy-Mediated DNA Sequencing
The DNA sequencing was performed by the dideoxy-mediated DNA sequencing technique of Sanger (13) using the Sequenase Kit (United States Biochemical Corporation, Cleveland, OH). The radioactive labeling reaction was carried out at 17°C using 5'-[a- 35S] deoxyandenosine triphosphate. After completion of the sequencing reactions, the product was either placed directly on the sequencing gel or was frozen at -70°C and used within 3 days.
merase. The experimental and control DNA sequences were compared with the previously published data (9). RESULTS
All 15 study patients and the normal male control were found to have exons two through eight intact by PCR analysis with each of the PCR products found to be the expected size and without evidence of major molecular deletions. A family of five affected complete AIS individuals were studied in further detail with attention placed on the androgenbinding domain (Fig. 2) (11,12). All of the patients including one obligate carrier and one potential carrier had a normal size PCR segment for exon five (Fig. 3). Exon five was then sequenced in both orientations in three affected individuals chosen at random within the family. All three of these related family members (labeled A, E, and F in Fig. 3) were found to have the same nucleotide substitution of guanine to adenine at nucleotide position 2293. Additional aliquots of genomic DNA were sequenced in each of the three family members, which confirmed the point mutation. This nucleotide substitution presumably resulted in a change in the messenger ribonucleic acid (mRNA) translation of the hAR from alanine765 to threonine765 (Fig. 4). DISCUSSION
Numerous publications have described other molecular alterations in exons four, six, and seven, all
DNA
Gel Electrophoresis and Autoradiography
Ladder A
Patients 8 C 0 E F G Control
The sequencing products were loaded on a 6% polyacrylamide sequencing electrophoretic gel (Life Technologies, Inc., Gaithersburg, MD). The electrophoresis was run for a total of 3 to 4 hours wherein the gel was dried. Autoradiography of the dried gel was carried out at room temperature for 48 to 72 hours. Analysis
The nucleotide sequences were deduced manually from the auto radiograph, and all areas in question, including possible point mutations, were also examined by sequencing the complementary strand. In addition, when a point mutation was found, the entire analysis was repeated starting with another genomic DNA sample from the same patient to rule out the potential placement of an aberrant nucleotide with subsequent amplification by the Taq polyVol. 58, No.4, October 1992
<-283 bp
Figure 3 The PCR of exon five of the hAR including flanking intron segments. The products are of equal and expected 283 bp size in family members A, B, C, D, E, F, G, and the normal fertile male control. Patient B is an obligate carrier, and patient D is a potential carrier, whereas the remainder are affected with complete androgen insensitivity. The 123 bp DNA ladder is used as a standard size marker.
Sweet et al.
Point mutation in the androgen receptor gene
705
C-termlnus
3'
A C G T
t
~~lpq
~C]Ala =(2293) Ii -
pq[~~ ====-=
165
Thr [ C A(2293)=
3=:==~] Phe
Phe
~~] Tyr
Tyr
~T Normal Male Control
nucleotide
amino acid
-
A C G T
[C_ ~ ___
l~~
C---------
-
Family Member A
nucleotide
5' N-terminus Figure 4 Partial nucleotide sequence of exon five of the hAR in a family with complete AIS, revealing a point mutation at nucleotide position 2293 with a guanine -+ adenine base alteration resulting in an alanine765 -+ threonine765 amino acid substitution.
located in the androgen-binding domain, in complete AIS individuals (9, 10, 11, 14-16). Molecular mutations in the hAR may result in alterations of the mRNA and hAR. Sai et al. (15) and Marcelli et al. (14) each reported nucleotide changes in exons four and six, respectively, in patients with complete AIS. Both of these alterations resulted in the creation of a stop codon with a truncated hAR resulting. RisStalpers et al. (16) detected a point mutation in a complete AIS patient at the exon/intron boundary of exon four. Presumably, the loss in the normal splicing region resulted in the activation of one of the cryptic splicing sites upstream, forming a truncated hAR. Single base substitutions may also result in a change in the polarity of the receptor. The work presented here describes a previously unreported point mutation in exon five that potentially results in the conversion of a nonpolar AA to a polar AA. It is understood that the exon organization and bp homology of the hAR is similar to the estrogen receptor (ER) and other steroid receptor genes (6). Tora et al. (17) detected a point mutation, an apparent cloning artifact, in an ER cDNA clone located in exon five. This single nucleotide alteration resulted in the incorporation of a nonpolar AA valine for a polar AA glycine, thereby altering the polarity of the ER and consequently causing a markedly decreased affinity for E. Tora et al. (17) further confirmed the finding with site-directed mutagenesis, which also resulted in the formation of an ER that bound E poorly. According to secondary structure predictions, it was believed that the AA conversion 706
Sweet et aI.
Point mutation in the androgen receptor gene
resulted in the destabilization of the a-helix-{3-turn{3-strand in the steroid-binding domain. The abnormality reported in this current study is very similar to the one reported by Tora et al. (17) in which a molecular alteration resulted in the incorporation of an AA with differing polarity. It is possible that a conformational change in the steroid-binding domain could influence the androgen-hAR binding, the unmasking of the DNA-binding domain or perhaps even change the effect that the androgen-hAR complex has on target genes. It is likely that the unique molecular alteration in exon five of the androgen-binding domain described in this current study resulted in the complete AIS presentation. Previous work has revealed that the only area of known polymorphism formation in the hAR gene is in exon one (14, 18, 19), whereas there is no evidence to date that there are any polymorphisms located in the highly conserved DNA or steroid-binding domains. In addition, finding the identical molecular substitution in three of three affected family members sequenced further diminishes the possibility that the molecular alteration is a polymorphism. Additional DNA samples were not available for analysis from other unaffected male family members. Further receptor studies are currently underway to add additional support to the potential androgen-hAR affinity changes. The information reported in this study adds to the growing body of data that examine the locations of molecular alterations in patients with AIS. A specific area of frequent mutations or deletions, if one actually exists, has yet to be determined. Continued Fertility and Sterility
examination of the nature of genetic defects of the hAR gene in androgen insensitive patients may aid in future family counseling, antenatal diagnosis, and the possible treatment of the affected AIS individuals. Acknowledgments. Special thanks are extended to Roger J. Byrd, Ph.D., Department of Cytogenetics, Medical College of Georgia, Augusta, Georgia, for the fibroblast cultures and karyotype analysis and to Michael Koutsilieris, M.D., Bioregulation Hormonale, Setfoy P.Q., Canada for ongoing androgen receptor analysis. We also thank the following individuals for their cooperation and assistance in obtaining patient blood samples: Mary S. Maddox, Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia; W. Glenn Hurt, M.D., Department of Obstetrics and Gynecology, Medical College of Virginia, Richmond, Virginia; Bobby Shull, M.D., Department of Obstetrics and Gynecology, Scott and White Memorial Hospital and Clinic, Temple, Texas; Peyton Taylor, M.D., Department of Obstetrics and Gynecology, University of Virginia Medical School, Charlottesville, Virginia; Marvin Swanson, M.D., and Marvin Yussman, M.D., Department of Obstetrics and Gynecology, University of Louisville, Louisville, Kentucky; and William Hardeman, M.D., Athens, Georgia.
REFERENCES 1. Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FW, Migeon CJ. Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome. Proc Nat! Acad Sci USA 1988;85:8151-5. 2. Brown CJ, Gross SJ, Lubahn DB, Joseph DR, Wilson EM, French FS, et al. Androgen receptor locus on the human X chromosome: regional localization to Xql1-12 and description of a DNA polymorphism. Am J Hum Genet 1989;44:264-9. 3. Migeon BR, Brown TR, Axelman J, Migeon CJ. Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse. Proc Nat! Acad Sci USA 1981;78:6339-43. 4. Chang C, Kokontis J, Liao S. Molecular cloning of human and rat complementary DNA encoding the androgen receptors. Science 1988;240:324-6. 5. Trapman J, Klaassen P, Kuiper GGJM, van der Korput JAGM, Faber PW, van Rooij HCJ, et al. Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Commun 1988;153:241-8. 6. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM. Cloning of the human androgen receptor complementary DNA and localization of the X chromosome. Science 1988;240:327-30.
Vol. 58, No.4, October 1992
7. Kuiper GGJM, Faber PW, van Rooij HCJ, van der Korput JAGM, Ris-Stalpers C, Klaassen P, et al. Structural organization of the human androgen receptor gene. J Mol EndocrinoI1989;2:Rl-4. 8. Brinkman AO, Faper PW, van Rooij HCJ, Kuiper GGJM, Ris C, Klaassen P, et al. The human androgen receptor: domain, structure, genomic organization and regulation of expression. J Steroid Biochem 1989;34:307-10. 9. Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, et al. Sequence of the intron/exonjunctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Nat! Acad Sci USA 1989;86: 9534-8. 10. Brown TR, Lubahn DB, Wilson EM, French FS, Migeon CJ, Corden JL. Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. Mol Endocrinol 1990;4:1759-72. 11. DiLauro SL, Behzadian A, Tho SPT, McDonough PG. Probing genomic deoxyribonucleic acid for gene rearrangement in 14 patients with androgen insensitivity syndrome. Fertil SteriI1991;55:481-5. 12. Shull BL, Taylor PT. Testicular feminization syndrome. A case study of four generations. South Med J 1989;82:251-4. 13. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Press, 1989. 14. Marcelli M, Tilley WD, Wilson CM, Wilson JD, Griffin JE, McPhaul MJ. A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance. J Clin Invest 1990;85:1522-7. 15. Sai T, Seino S, Chang C, Trifiro M, Pinsky L, Mhatre A, et al. An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet 1990;46:1095-1100. 16. Ris-Stalpers C, Kuiper GGJM, Faper PW, Schweikert HU, van Rooij HCJ, Zegers ND, et al. Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity. Proc Natl Acad Sci USA 1990;87:7866-70. 17. Tora L, Mullick A, Metzfer D, Ponglikitmongkol M, Park I, Chambon P. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J 1989;8:1981-6. 18. Lubahn DB, Joseph DR, Sar FM, Tan J, Higgs HN, Larson RE, et al. The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol EndocrinoI1988;2:1265-75. 19. Faber PW, Kuiper GGJM, van Rooij HCJ, van der Korput JAGM, Brinkmann AO, Trapman J. The N-terminal domain of the human androgen receptor is encoded by one large exon. Mol Cell Endocrinol 1989;61:257-62.
Sweet et al.
Point mutation in the androgen receptor gene
707
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright 4:) 1992 The American Fertility Society
A unique point mutation in the androgen receptor gene in a family with complete androgen insensitivity syndrome*
Craig R. Sweet, M.D.t;!: Mohammad A. Behzadian, Ph.D.t§ Paul G. McDonough, M.D.tll Medical College of Georgia, Augusta, Georgia
Objective: To further delineate the diversity of genetic alterations in the gene coding for the androgen receptor in individuals with the androgen insensitivity syndrome and to increase our understanding of the disease at the molecular level. Design: This was a prospective study in which genomic deoxyribonucleic acid (DNA) from individuals with androgen insensitivity were examined through the polymerase chain reaction and DNA sequencing analysis. Patients: Eleven complete and four individuals with partial androgen insensitivity syndrome were examined. Results: Exons two through eight were grossly intact in all study subjects. Nucleotide sequence analysis revealed that three of three related family members with complete androgen insensitivity had the same guanine to adenine base substitution in exon five of the steroid-binding domain. Conclusion: The subsequent alanine to threonine amino acid conversion may have resulted in a configurational change of the androgen receptor protein leading to complete androgen insensitivity. This precise alteration has not been previously identified in the human androgen receptor gene in patients with the androgen insensitivity syndrome. Fertil Steril 1992;58:703-7 Key Words: Androgen insensitivity syndrome, point mutation
Androgens act through the human androgen receptor (hAR) to direct sexual differentiation and maintenance of the male phenotype with normal testicular exocrine function. Genetic rearrangements in the hAR gene may be responsible for a wide spectrum of functional abnormalities in the hAR resulting in the androgen insensitivity syndrome (AlS) (1). These abnormalities range from complete AlS
Received February 25, 1992; revised and accepted June 19, 1992. * Presented at the 38th Annual Meeting of the Society of Gynecologic Investigation, San Antonio, Texas, March 20 to 23, 1991. t Department of Obstetrics and Gynecology. :j: Present address: Women's Health Care and Reproductive Services, Fort Myers, Florida. § Department of Anatomy. II Reprint requests: Paul G. McDonough, M.D., Division ofReproductive Endocrinology, Medical College of Georgia, Augusta, Georgia 30912-3360. Vol. 58, No.4, October 1992
wherein 46,XY phenotypic females have a blind vaginal pouch and intra-abdominal or inguinal testes to partial AlS with genital ambiguity, hypospadias, or defects in spermatogenesis with azoospermia. The localization of the gene for the hAR to Xql1-12(2, 3) and the isolation of the complementary deoxyribonucleic acid (cDNA) in 1988 set the stage to study the molecular pathology of AlS in humans (4-6). The hAR gene, including introns, is >90 kb in length, although the coding region is divided into eight exons that are <3 kb in combined length (Fig. 1) (7-9). The hAR binds with androgens forming the androgen-hAR complex. This complex then binds to specific target genes that are responsible for the expression of the male phenotype and reproductive capabilities. Exon one codes for the N-terminal hAR domain and is the largest of the eight exons. It is hypothesized that the N-terminus Sweet et a1.
Point mutation in the androgen receptor gene
703
hAR Gene > 90 kb
I
I
•
I II eDNA 2757 bp
c
Amino Terminus Domain 1613 bp
Exon1
2345678
DNA Binding Domain 269bp
I
Steroid Binding Domain 875 bp
Protein 619 AA
Figure 1 Schematic representation of the X -linked hAR gene organization.
modulates transcription once the androgen-hAR complex is bound to the target gene DNA (9). The highly conserved DNA-binding domain codes for the two zinc finger motifs and is located in exons two and three. These areas may be involved in the recognition of the target genes in addition to stabilizing the androgen-hAR-DNA interaction (7). The remaining exons four through eight code for the androgen-binding domain. This region of the hAR gene may also be involved in unmasking the DNA-binding domain or the transcriptional activation of the target genes (8, 10). The aim of this research was to further characterize the molecular rearrangements in the hAR gene and expand our knowledge of the spectrum of molecular alterations responsible for AIS. MATERIALS AND METHODS Study Subjects
This research was approved by the Human Assurance Committee of the Medical College of Georgia. Informed consent was obtained in accordance with the Medical College of Georgia guidelines for Clinical Investigation. The subject population included sporadic (n = 6) and a family (n = 5) of complete AIS patients in addition to a sporadic (n = 1) and a family (n = 3) partial AIS individuals for a total study population of 15. The complete AIS family, shown in Figure 2, has been previously discussed (11, 12). A normal fertile male and the published normal DNA sequence served as controls (9). First-degree relatives were also examined when available and indicated. The complete AIS patients were characterized by 46,XY G-banded karyotypes, normal female external genitalia, and breast development, a blind vaginal pouch, sparse axillary and pubic hair, intra-abdominal or inguinal testes, and blood testosterone con704
Sweet et al.
Point mutation in the androgen receptor gene
centrations in the normal male range or greater. The partial AIS patients had 46,XY G-banded karyotypes with genital ambiguity and intra-abdominal or labioscrotal testes with a quantitative or qualitative deficiency in their hAR-androgen binding. Preparation of Genomic DNA and Oligonucleotide Primers
The genomic DNA was isolated from leukocytes, transformed leukocytes, or fibroblast cells (13). For each exon two through eight, two oligonucleotide primers were specifically designed to anneal to the flanking intron segments and were synthesized on the DNA synthesizer Gene Assembler Plus (Phamacia, Piscataway, NJ). The primers were 22 to 29 nucleotides in length and were similar to those used by Lubahn et al. (9). Polymerase Chain Reaction (PCR)
Polymerase chain reactions were performed in a final volume of 100 tiL containing buffer, 0.1 tIg of genomic DNA, 1.5 tiM MgCI2 , 200 tiM of each deoxynucleotide triphosphate (2'-deoxyadenosine 5'-triphosphate, 2'-deoxycytidine 5'-triphosphate, 2'deoxyguanosine 5'-triphosphate, and 2'-deoxythymidine 5'-triphosphate), 1.5 U of Taq DNA polymerase, and 1.0 tiM of each flanking primer (GeneAmp Kit; Perkin-Elmer/Cetus Instruments, Norwalk, CT). The PCR reaction was performed in a DNA Thermal Cycler (Perkin-Elmer/Cetus Instruments). The DNA was first denatured at 96°C, the primers annealed at 55 to 58°C, depending on the exon being examined, and the DNA extension was achieved at 72°C; each of these steps lasted 5 minutes. The reaction then continued at the same temperatures for 1 minute at each step for a total
Figure 2 Pedigree of the complete AIS family showing X-linked inheritance pattern. Family members A, B, C, D, E, F, and G were studied with PCR and family members A, E, and F undergoing nucleotide sequence analysis. Il!il are affected 46,XY; II!i are affected and sequenced in both orientations; 0 are obligate heterozygotes 46,XX (11). (Reprinted by permission of the publisher, The American Fertility Society, Birmingham, AL.)
Fertility and Sterility
of 30 cycles. At the completion of the 30 cycles, an additional 5-minute incubation at 72°C allowed for the completion of partially synthesized strands. The PCR products were examined on an electrophoretic agarose gel, and the respective lengths of the segments estimated using known DNA size markers (Bethesda Research Laboratories, Gaithersburg, MD). These segments ranged from 250 to 480 base pairs (bps) in length and were compared with the normal fertile male control and previously published data (9). Asymmetric PCR
The asymmetric PCR was performed to synthesize single-stranded DNA (ssDNA) for nucleotide sequence analysis. An aliquot of 1 ILL of the first PCR product was added to only a single primer and other constituents as described with the symmetric PCR. Fifty PCR cycles were performed to maximize the synthesis of the ssDNA. Dideoxy-Mediated DNA Sequencing
The DNA sequencing was performed by the dideoxy-mediated DNA sequencing technique of Sanger (13) using the Sequenase Kit (United States Biochemical Corporation, Cleveland, OH). The radioactive labeling reaction was carried out at 17°C using 5'-[a- 35S] deoxyandenosine triphosphate. After completion of the sequencing reactions, the product was either placed directly on the sequencing gel or was frozen at -70°C and used within 3 days.
merase. The experimental and control DNA sequences were compared with the previously published data (9). RESULTS
All 15 study patients and the normal male control were found to have exons two through eight intact by PCR analysis with each of the PCR products found to be the expected size and without evidence of major molecular deletions. A family of five affected complete AIS individuals were studied in further detail with attention placed on the androgenbinding domain (Fig. 2) (11,12). All of the patients including one obligate carrier and one potential carrier had a normal size PCR segment for exon five (Fig. 3). Exon five was then sequenced in both orientations in three affected individuals chosen at random within the family. All three of these related family members (labeled A, E, and F in Fig. 3) were found to have the same nucleotide substitution of guanine to adenine at nucleotide position 2293. Additional aliquots of genomic DNA were sequenced in each of the three family members, which confirmed the point mutation. This nucleotide substitution presumably resulted in a change in the messenger ribonucleic acid (mRNA) translation of the hAR from alanine765 to threonine765 (Fig. 4). DISCUSSION
Numerous publications have described other molecular alterations in exons four, six, and seven, all
DNA
Gel Electrophoresis and Autoradiography
Ladder A
Patients 8 C 0 E F G Control
The sequencing products were loaded on a 6% polyacrylamide sequencing electrophoretic gel (Life Technologies, Inc., Gaithersburg, MD). The electrophoresis was run for a total of 3 to 4 hours wherein the gel was dried. Autoradiography of the dried gel was carried out at room temperature for 48 to 72 hours. Analysis
The nucleotide sequences were deduced manually from the auto radiograph, and all areas in question, including possible point mutations, were also examined by sequencing the complementary strand. In addition, when a point mutation was found, the entire analysis was repeated starting with another genomic DNA sample from the same patient to rule out the potential placement of an aberrant nucleotide with subsequent amplification by the Taq polyVol. 58, No.4, October 1992
<-283 bp
Figure 3 The PCR of exon five of the hAR including flanking intron segments. The products are of equal and expected 283 bp size in family members A, B, C, D, E, F, G, and the normal fertile male control. Patient B is an obligate carrier, and patient D is a potential carrier, whereas the remainder are affected with complete androgen insensitivity. The 123 bp DNA ladder is used as a standard size marker.
Sweet et al.
Point mutation in the androgen receptor gene
705
C-termlnus
3'
A C G T
t
~~lpq
~C]Ala =(2293) Ii -
pq[~~ ====-=
165
Thr [ C A(2293)=
3=:==~] Phe
Phe
~~] Tyr
Tyr
~T Normal Male Control
nucleotide
amino acid
-
A C G T
[C_ ~ ___
l~~
C---------
-
Family Member A
nucleotide
5' N-terminus Figure 4 Partial nucleotide sequence of exon five of the hAR in a family with complete AIS, revealing a point mutation at nucleotide position 2293 with a guanine -+ adenine base alteration resulting in an alanine765 -+ threonine765 amino acid substitution.
located in the androgen-binding domain, in complete AIS individuals (9, 10, 11, 14-16). Molecular mutations in the hAR may result in alterations of the mRNA and hAR. Sai et al. (15) and Marcelli et al. (14) each reported nucleotide changes in exons four and six, respectively, in patients with complete AIS. Both of these alterations resulted in the creation of a stop codon with a truncated hAR resulting. RisStalpers et al. (16) detected a point mutation in a complete AIS patient at the exon/intron boundary of exon four. Presumably, the loss in the normal splicing region resulted in the activation of one of the cryptic splicing sites upstream, forming a truncated hAR. Single base substitutions may also result in a change in the polarity of the receptor. The work presented here describes a previously unreported point mutation in exon five that potentially results in the conversion of a nonpolar AA to a polar AA. It is understood that the exon organization and bp homology of the hAR is similar to the estrogen receptor (ER) and other steroid receptor genes (6). Tora et al. (17) detected a point mutation, an apparent cloning artifact, in an ER cDNA clone located in exon five. This single nucleotide alteration resulted in the incorporation of a nonpolar AA valine for a polar AA glycine, thereby altering the polarity of the ER and consequently causing a markedly decreased affinity for E. Tora et al. (17) further confirmed the finding with site-directed mutagenesis, which also resulted in the formation of an ER that bound E poorly. According to secondary structure predictions, it was believed that the AA conversion 706
Sweet et aI.
Point mutation in the androgen receptor gene
resulted in the destabilization of the a-helix-{3-turn{3-strand in the steroid-binding domain. The abnormality reported in this current study is very similar to the one reported by Tora et al. (17) in which a molecular alteration resulted in the incorporation of an AA with differing polarity. It is possible that a conformational change in the steroid-binding domain could influence the androgen-hAR binding, the unmasking of the DNA-binding domain or perhaps even change the effect that the androgen-hAR complex has on target genes. It is likely that the unique molecular alteration in exon five of the androgen-binding domain described in this current study resulted in the complete AIS presentation. Previous work has revealed that the only area of known polymorphism formation in the hAR gene is in exon one (14, 18, 19), whereas there is no evidence to date that there are any polymorphisms located in the highly conserved DNA or steroid-binding domains. In addition, finding the identical molecular substitution in three of three affected family members sequenced further diminishes the possibility that the molecular alteration is a polymorphism. Additional DNA samples were not available for analysis from other unaffected male family members. Further receptor studies are currently underway to add additional support to the potential androgen-hAR affinity changes. The information reported in this study adds to the growing body of data that examine the locations of molecular alterations in patients with AIS. A specific area of frequent mutations or deletions, if one actually exists, has yet to be determined. Continued Fertility and Sterility
examination of the nature of genetic defects of the hAR gene in androgen insensitive patients may aid in future family counseling, antenatal diagnosis, and the possible treatment of the affected AIS individuals. Acknowledgments. Special thanks are extended to Roger J. Byrd, Ph.D., Department of Cytogenetics, Medical College of Georgia, Augusta, Georgia, for the fibroblast cultures and karyotype analysis and to Michael Koutsilieris, M.D., Bioregulation Hormonale, Setfoy P.Q., Canada for ongoing androgen receptor analysis. We also thank the following individuals for their cooperation and assistance in obtaining patient blood samples: Mary S. Maddox, Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia; W. Glenn Hurt, M.D., Department of Obstetrics and Gynecology, Medical College of Virginia, Richmond, Virginia; Bobby Shull, M.D., Department of Obstetrics and Gynecology, Scott and White Memorial Hospital and Clinic, Temple, Texas; Peyton Taylor, M.D., Department of Obstetrics and Gynecology, University of Virginia Medical School, Charlottesville, Virginia; Marvin Swanson, M.D., and Marvin Yussman, M.D., Department of Obstetrics and Gynecology, University of Louisville, Louisville, Kentucky; and William Hardeman, M.D., Athens, Georgia.
REFERENCES 1. Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FW, Migeon CJ. Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome. Proc Nat! Acad Sci USA 1988;85:8151-5. 2. Brown CJ, Gross SJ, Lubahn DB, Joseph DR, Wilson EM, French FS, et al. Androgen receptor locus on the human X chromosome: regional localization to Xql1-12 and description of a DNA polymorphism. Am J Hum Genet 1989;44:264-9. 3. Migeon BR, Brown TR, Axelman J, Migeon CJ. Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse. Proc Nat! Acad Sci USA 1981;78:6339-43. 4. Chang C, Kokontis J, Liao S. Molecular cloning of human and rat complementary DNA encoding the androgen receptors. Science 1988;240:324-6. 5. Trapman J, Klaassen P, Kuiper GGJM, van der Korput JAGM, Faber PW, van Rooij HCJ, et al. Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Commun 1988;153:241-8. 6. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM. Cloning of the human androgen receptor complementary DNA and localization of the X chromosome. Science 1988;240:327-30.
Vol. 58, No.4, October 1992
7. Kuiper GGJM, Faber PW, van Rooij HCJ, van der Korput JAGM, Ris-Stalpers C, Klaassen P, et al. Structural organization of the human androgen receptor gene. J Mol EndocrinoI1989;2:Rl-4. 8. Brinkman AO, Faper PW, van Rooij HCJ, Kuiper GGJM, Ris C, Klaassen P, et al. The human androgen receptor: domain, structure, genomic organization and regulation of expression. J Steroid Biochem 1989;34:307-10. 9. Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, et al. Sequence of the intron/exonjunctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Nat! Acad Sci USA 1989;86: 9534-8. 10. Brown TR, Lubahn DB, Wilson EM, French FS, Migeon CJ, Corden JL. Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. Mol Endocrinol 1990;4:1759-72. 11. DiLauro SL, Behzadian A, Tho SPT, McDonough PG. Probing genomic deoxyribonucleic acid for gene rearrangement in 14 patients with androgen insensitivity syndrome. Fertil SteriI1991;55:481-5. 12. Shull BL, Taylor PT. Testicular feminization syndrome. A case study of four generations. South Med J 1989;82:251-4. 13. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Press, 1989. 14. Marcelli M, Tilley WD, Wilson CM, Wilson JD, Griffin JE, McPhaul MJ. A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance. J Clin Invest 1990;85:1522-7. 15. Sai T, Seino S, Chang C, Trifiro M, Pinsky L, Mhatre A, et al. An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet 1990;46:1095-1100. 16. Ris-Stalpers C, Kuiper GGJM, Faper PW, Schweikert HU, van Rooij HCJ, Zegers ND, et al. Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity. Proc Natl Acad Sci USA 1990;87:7866-70. 17. Tora L, Mullick A, Metzfer D, Ponglikitmongkol M, Park I, Chambon P. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J 1989;8:1981-6. 18. Lubahn DB, Joseph DR, Sar FM, Tan J, Higgs HN, Larson RE, et al. The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol EndocrinoI1988;2:1265-75. 19. Faber PW, Kuiper GGJM, van Rooij HCJ, van der Korput JAGM, Brinkmann AO, Trapman J. The N-terminal domain of the human androgen receptor is encoded by one large exon. Mol Cell Endocrinol 1989;61:257-62.
Sweet et al.
Point mutation in the androgen receptor gene
707